GSM5070305: ChIP Rad21 30min control rep1; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
RAD21
Cell type
Cell type Class
Uterus
Cell type
HeLa
Primary Tissue
Cervix
Tissue Diagnosis
Adenocarcinoma
Attributes by original data submitter
Sample
source_name
HeLa cells
cell line
HeLa
cell type
cervical carcinoma cells
chip antibody
Rad21 (Abcam, ab992)
treatment
isotonic buffer
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
C-TALE was performed as described previously (Golov AK, Ulianov SV, Luzhin AV, Kalabusheva EP, Kantidze OL, Flyamer IM, Razin SV, Gavrilov AA. C-TALE, a new cost-effective method for targeted enrichment of Hi-C/3C-seq libraries. Methods. 2020 Jan). For ChIP-seq experiments, fixed chromatin pellets were resuspended in 600 uL of ice cold RIPA buffer supplemented with protease inhibitors. Chromatin was sheared on ice with a VirSonic 100 cell disrupter with 10 30-sec pulses on “15” power setting, separated with 3 min periods of recovery. Input DNA was isolated from 1/10 aliquots of sonicated samples. Sheared chromatin from approximately 1 mln of cells and 1 ug of antibodies (against either CTCF, Smc3, or Rad21) were used in each immunoprecipitation reaction. Chromatin immunoprecipitation was performed as described in Abcam X-ChIP manual (https://www.abcam.com/protocols/cross-linking-chromatin-immunoprecipitation-x-chip-protocol) with minor modifications. 25 ul of protein A/G magnetic beads (Thermo Scientific, 26162) was used per reaction instead of agarose beads, thus a magnet was used instead of a centrifuge to reclaim beads. Beads were blocked with 1% BSA solution in RIPA overnight, while chromatin was incubated with antibodies. BSA-blocked beads were mixed with antibody-bound chromatin for 6 h. Immunoprecipitated DNA was separated from beads by overnight treatment with proteinase K (Thermo Scientific, EO0491) in PBS supplemented with 1% SDS at 65C. Solution was then cleared from beads with a magnet and DNA was isolated with standard phenol-chloroform extraction and ethanol precipitation. Sequencing libraries from 3C-seq material, immunoprecipitated DNA and ChIP inputs were generated as described previously (Golov AK, Ulianov SV, Luzhin AV, Kalabusheva EP, Kantidze OL, Flyamer IM, Razin SV, Gavrilov AA. C-TALE, a new cost-effective method for targeted enrichment of Hi-C/3C-seq libraries. Methods. 2020 Jan).